The intracellular redox balance is crucial for maintaining normal physiological functions, while the delicate equilibrium between oxidative stress and antioxidant mechanisms plays a pivotal role in cellular health [1]. In recent years, researches have been focused on ferroptosis, an iron-dependent form of cell death [2,3]. Ferroptosis is closely associated with the abnormal accumulation of intracellular iron and oxidative stress, as well as its critical connection to antioxidant redox systems [4,5]. These intracellular antioxidant systems mainly include glutathione (GSH) and thioredoxin (Trx) systems, both are substrates of glutathione peroxide 4 (GPX4), which help to preserve the intracellular redox balance and mitigates cellular oxidative stress [6,7]. Importantly, current researches suggest that most ferroptosis inducers primarily act by interfering with the GSH system [8]. For example, Erastin, a classic ferroptosis inducer, directly inhibits system Xc- activity leading to the reduction of GSH synthesis, thus the subsequent depletion of GPX4, ultimately resulting in ferroptosis [9]. Additionally, studies have identified other ferroptosis inducers such as Auranofin [10] that disrupt ferroptosis through inhibition of thioredoxin reductase (TrxRD), causing lipid peroxide accumulation in cell membranes and eventual cell death. It is worth noting that the modulation of a single antioxidant system to induce ferroptosis can trigger compensatory upregulation of other antioxidant systems, which can potentially reduce the overall effectiveness of ferroptosis induction [11].

Indeed, both ferroptosis and antioxidant systems are under the influence of nuclear factor erythroid 2-related factor 2 (Nrf2) [12], a pivotal cellular regulator of the oxidative stress response. Activation of Nrf2 can initiate the expression of a cascade of antioxidant genes, including glutathione reductase, glutathione S-transferase, Trx, and others, thereby enhancing the cell's defense against oxidative damage, overexpression of Nrf2 further increases the antioxidant and puts it in a reduced state [13]. Furthermore, it was found that GPX4 expression could be directly decreased by inhibiting Nrf2, leading to impairment of intracellular antioxidant systems; or regulation of iron-related genes for controlling intracellular iron distribution and accumulation, ultimately resulting in ferroptosis [14]. For example, Zhang et al. [15] found that Baicalin was involved in ferroptosis by inhibiting the axis Nrf2/Solute Carrier Family 7 Member 11 (SLC7A11/xCT)/GPX4. The combination of the GSH antioxidant system inhibitor sulfoximine (BSO) with the Trx antioxidant system inhibitors sulfasalazine (SSA) or auranofin (AUR) has been reported to significantly enhance the antitumor efficacy, but it also introduces toxic side effects [16]. However, no ferroptosis inducers have been discovered that modulate both GSH and Trx dual antioxidant redox systems through inhibiting Nrf2.

Gambogic acid (GA) is the main active compound in traditional Chinese medicine Gamboge. Multiple studies have demonstrated that GA possesses anti-cancer potential through various molecular mechanisms, including induce of apoptosis, autophagy, cell cycle arrest, as well as the inhibition of cell invasion, metastasis, and angiogenesis [17]. In our previous research, GA enhanced intracellular reactive oxygen species (ROS) production by depleting GSH [18]. Remarkably, literatures suggested that GA could interfere with TrxRD activity, resulting in ROS accumulation and disruption of intracellular redox balance [19]. This intriguing finding prompts us to hypothesize that GA may induce ferroptosis by modulating the antioxidant redox systems. However, the underlying mechanisms of this process require further elucidation.

In this study, Cell Counting Kit-8 (CCK8), colony formation assay and cell cycle assay were analyzed potent tumor-killing ability of GA on MCF-7 cells and HepG2 cells in vitro. Cell death type of GA was established by co-incubation with inhibitors and RNA sequencing. Ferroptosis indicators detection was analyzed confocal laser scanning microscopy (CLSM), flow cytometry and transmission electron microscopy (TEM). Western Blot (WB), PCR and molecular docking were used to confirm the mechanism of GA-induced ferroptosis. Subcutaneous tumor model with MCF-7 cells was constructed to evaluate the efficacy of GA, and the related proteins and ferroptosis indicators were investigated by immunohistochemical (IHC), fluorescence staining and CLSM imaging. In conclusion, our results support the assumption that GA can induce ferroptosis through inhibiting Nrf2-mediated dual antioxidant system comprising GSH and Trx.